Dexmedetomidine modifies uterine contractions in pregnancy terms of rats.

OBJECTIVES: The present study was aimed at determining the effective doses of Dexmedetomidine (Dex) involved in amplitude of contraction-force and frequency of uterine rings in pregnancy terms of rats. All experiments involving animal subjects were carried out with the approval of animal care and use Ethical Committee of Cukurova University. Experiments were performed on female Albino-Wistar rats (200-260 g; n = 40).
MATERIALS AND METHODS: Uterine rings from pregnant rats were placed in organ bath with Krebs and calcium ion (Ca(2+))-free solutions to record and exposed to serially increasing log10 concentrations of Dex.
RESULTS: In Krebs solution, while Dex caused an increase in the spontaneous contraction-forces in all pregnancy terms of rats in a significant dose-dependent manner, it led to a decrease in contraction-frequency in late-pregnancy term of rats. In Ca(2+)-free, the spontaneous contraction-force decreased in late-pregnancy term and increased in early and middle-pregnancy terms. In addition, while Dex increased the contraction-frequency in early and middle-pregnancy terms, it decreased in late-pregnancy term in a dose-dependent manner. STATISTICAL ANALYSIS USED: The data were subjected to one-way analysis of variance. Repeated measures were employed for comparison of several group means through the Tukey post-hoc test (SPSS 10.00 Inc., Chicago, Ill, USA). P < 0.05 was considered statistically significant.
CONCLUSIONS: This study suggested that Dex might differently alter the spontaneous contraction-forces and contraction-frequencies of uterine rings in all pregnancy terms of rats in Krebs and Ca(2+)-free solutions.

Introduction

Contractions of the myometrial smooth muscle cells comprising most of the myometrial layer of the uterine wall bring about uterine contractility.[1] The contractile response of smooth muscle is essentially triggered by an increase in calcium ion (Ca2+) concentration. Not only the influx of Ca2+, but also the release of stored Ca2+ raises receptor agonists, such as oxytocin which elevates in pregnancy terms.[2-8]

Ca2+ entry into the cells through voltage-operated Ca2+ channels is initiated by the depolarization of the cell membrane initiates, contributing to a rise in the intracellular Ca2+ concentration (Ca,i) and muscle contraction.[9] Calcium entry from the extracellular environment can make a contribution in a significant way to the total intracellular calcium-free pool and to the refilling of intracellular stores, as well.

Calcium entry can be regulated through several mechanisms including activation of receptor-operated and second-messenger-operated cation channels, L-type voltage-operated calcium channels, and calcium release-activated calcium entry which as a consequence of depletion of intracellular stores.[10-12] Modulation of the activity of potassium and other ion channels can potentially affect calcium homeostasis through influences on membrane potential. As it is well known, whereas α1-adrenergic agonists conduce contractility, β2-adrenergic agonists prevent it; the myometrial β2-adrenoceptors elevate the intracellular cyclic adenosine monophosphate (cAMP) level and lead to uterine relaxation[13] while the α-adrenergic receptors (α1, α2- ARs) agonists elicit contractions via increases in the intracellular inositol phosphate and Ca2+ levels.[14]

However, α2-agonists can activate the α1-adrenoreceptors; hence, activation of α1-adrenoreceptors triggers arousal, restlessness, increased locomotor activity, and elevated vigilance. Therefore, the roles of the α2-ARs on myometrial contractility are not fully clear. Previous studies indicated that three subtypes of α2-ARs, termed α2A, α2B, and α2C, were initially revealed by pharmacological means and were cloned from several species, including animals and humans. Notwithstanding structural similarity and general conservation among mammalian species, there is a differential distribution of the α2-AR subtypes in cells and tissues, with different pharmacological and physiological profiles. As opposed to the pre-synaptic location of the α2A- and α2C adrenergic receptors, α2B-ARs are found chiefly post-synaptically.[1516] α2B-subtype seems to take a fundamental role in eliciting the vasoconstrictor response to α2-adrenergic agonists in vascular smooth muscle.[17] Furthermore, the other classified adrenoceptors in the myometrium, the selective distribution of α and β adrenoceptors sites, respectively, in circular or longitudinal layer support that endogenous catecholamines are excitatory in the circular muscle and inhibitory in the longitudinal muscle during pregnancy; however, the functional roles of the longitudinal and circular muscles is yet to be established.[18-22] Clinically, α2-adrenergic receptor agonists have been used extensively in the field of anesthesia. Dex is a recently developed-highly specific α2-AR agonist with a high affinity to each of the α2-AR subtypes, and it is eight times more specific to α2-ARs than clonidine in binding studies. We hypothesized that the response of α2-adrenergic receptor to Dex might be changeable in pregnancy terms. The aim of this study was to determine the effective doses of Dex involved in amplitude of contraction-force and contraction-frequency of isolated uterine rings in early, middle, and late-pregnancy terms of rats.

Materials and Methods

Housing and Handling of the Animals

All experiments involving animal subjects were carried out with the approval of animal care and use Ethical Committee of Cukurova University. Experiments were performed on female Albino-Wistar rats (200-260 g; n = 40). Albino-Wistar female rats were kept at 22 ± 3°C; the relative humidity was 50-60% and the light/dark cycle was 12/12 h. They were maintained on a standard pellet diet with tap water available ad libitum.

Mating of the Animals

Mature female (200-260 g) and male (240-260 g) rats were mated in a special mating cage. A female rat was mated with a male overnight to obtain a pregnant rat. The next morning, if a copulation plug was detected, was designated as day 1 of pregnancy. Then, pregnant rats were separated into three groups; Early pregnancy term of rats (n = 10), middle pregnancy term of rats (n = 10) and late pregnancy term of rats (n =10). Early term pregnant rats were used on days 7-8 of pregnancy, middle-term pregnant rats were used on days 14- 15 of pregnancy and late term pregnant rats were used on days 19-21 of pregnancy. Age- and weight-matched virgin rats (n = 10) were used as controls. The animals were killed by cervical dislocation. The entire uterus was rapidly excised, and fetuses and placentas were removed. The tissue was immediately placed in Kreb's solution.

Isolated Organ Bath Studies

3-4-mm-long uterine rings were sliced from the uterine horns and mounted in an organ bath (4 parallels) containing 20 ml Krebs solution (in mmol/L): NaCl 117; KCl 4.7; KH2 PO4 0.9; CaCl2 2.5; NaHCO3 25; glucose 10.1; sodium EDTA 0.03,) and Ca2+ -free solution, bubbled with 5 % carbon dioxide and 95 oxygen % in air and maintained at 37°C and a pH of approximately 7.40. After being mounted in organ baths (four organ baths), each uterine ring was allowed to equilibrate at 1.5 g tension for 60 min, after which the preparation was challenged twice by administration of a maximally effective concentration of oxytocin (1 mU/mL). Uterine rings were then allowed to equilibrate for a further 60 min and, when the contractions became regular, they were exposed to increasing log10 concentrations of the α2 -agonist Dex. Following the addition of each concentration of Dex, recording was performed for 20 min. Uterine strips in virgin rats were induced by oxytocin (1mU/mL) as pregnant rat model and the experimental procedure were repeated for virgin rats. Then, organ bath solutions were exchanged by Ca2+ -free solution and same experimental procedure was repeated for Ca2+ -free solution. The amplitude of contraction-force in uterine rings was measured with isometric force transducers (FDT 10A, Commat Ltd., Ankara, Turkey) and recorded by an MP35 Data Acquisition System (Biopac System, USA) [Figure 1].

Figure 1

(a) Diagram of isolated organ bath system: Isolated organ bath system having a glass Jacketed organ bath, a programmable heating circulator, FDT-10A displacement force transducer, a Biopac MP35 recording system and a computer. Muscle rings (3-4 mm long) were sliced from the uterine horns and mounted vertically in an organ bath containing 20 ml Krebs solution. The organ bath was maintained at 37°C with a circulator. Uterine contractile activity (force and frequency) were measured with a force-displacement (FDT-10A MAYCOM-Ankara, Turkey) and a Data Acquisition System (Biopac MP35 System), (b) an example contractile-force record obtained from uterine ring in early-term of pregnant rats

Drugs

Drug-containing solutions were prepared immediately before each experiment was performed. Precedex 200 μg/2 ml (Dexmedetomidine) (Dex) (Abbott Laboratories Inc., North Chicago, Ill, USA) was dissolved in distilled water and was prepared at concentrations of 10-9, 10-8, 10-7, 10-6, 10-5, and 10-4 mol/L and 0.1 mL of each concentration was added to 20 mL Krebs-buffer solution respectively. Oxytocin (oxytocin; Pitocin; Parke-Davis) was added directly to the organ baths so as to induce uterine rings of virgin rats. Nifedipine (Sigma) was dissolved in DMSO, and 10-5 mol/L concentration of nifedipine was added to the organ baths in volume of 0.1 mL.

Statistical Analysis

At the start of each test of experimental procedures, amplitude and frequency of spontaneous uterine contractions in all pregnancy terms of rats were considered as a reference response. The characteristics of contraction-force and frequency analyzed immediately before and after drugs were added (last 10 min) included amplitude and frequency. The amplitude of contraction-force was expressed as gram (g) and the changes in the number of contraction-frequencies were expressed as min-1. Data were presented as mean ± SEM and changes in the amplitude of (g) and frequency (per min-1) of spontaneous uterine contractions were expressed as percentages of the initial reference response and analyzed through one-way analysis of variance. Repeated measures were employed for comparison of several group means through the Tukey post-hoc test (SPSS 10.00 Inc., Chicago, Ill, USA). P < 0.05 was considered statistically significant.

Results

Isolated uterine rings from virgin rats in control group were induced by oxytocin. Then, increasing cumulative concentrations of Dex were added in organ baths. No significant differences between oxytocin and Dex-induced contraction-forces and contraction-frequencies were observed in control group (data not shown) (P > 0.05).

Effect of Dexmedetomidine on Spontaneous Contraction-forces

During pregnancy terms of rats (early, middle and late-terms of pregnancy), there were differences among the three groups with respect to the amplitudes of spontaneous contraction-force values or control values (3.57 ± 0.2 g, 4.98 ± 0.6 g and 10.49 ± 0.3 g, respectively) [Figure 2a]. After increasing concentration of Dex was added in organ baths, the uterine spontaneous contractions-forces increased in all pregnant groups in a concentration-dependent manner.

Figure 2

(a) Effects of calcium on Dexmedetomidine (Dex) -induced uterine contractile-force in pregnancy terms of pregnant rats. Dex was significantly changed the contractile-force in a concentration dependent manner in all pregnancy terms (early-term (●), middle-term (○) and late-term (▼). When compared the pregnant terms, Dex induced changes in contractile-force were also different. Each point represents the percentage mean value ± SEM. *P < 0.05 as compared to control at each concentration. #P < 0.05 as compared to late or early terms at each concentration, (b) Effects of calcium on Dex-induced uterine contractile-frequency in terms of pregnant rats. Dex changed the frequency of contraction-force in all pregnancy terms (early-term (●), middle-term (○) and late-term (▼). Each point represents the percentage mean value ± SEM. *P < 0.05 as compared to control at each concentration. #P < 0.05 as compared to early term at each concentration. +P < 0.05 as compared to early term at each concentration

While Dex in a dose of 10-9 mol/L decreased the percentage of the uterine contraction-forces in early-pregnancy term of rats by 97.2%, it increased the percentage of the uterine contraction-forces in middle pregnancy term of rats. However, after 10-8 M concentration of Dex was added in organ baths, the contraction-forces increased in all groups in a dose-dependent manner [Figure 2a].

Similarly, while Dex in a dose of 10-4 mol/L increased the percentage of contraction-force in early- and late pregnancy terms of rats [Figure 2a], it differently increased the percentage of contraction-force in uterine contraction in middle pregnancy term of rats in comparison to the percentage of contraction- force in early and late pregnancy terms of rats [Figure 2a].

Effect of Ca2+-Free Solution on Dexmedetomidine-Induced Contraction

When the Kreb's solution in organ baths was exchanged with Ca2+ -free solution, the baseline of spontaneous contraction-forces was decreased in all pregnant groups [Figure 2b] and this decrease in amplitude of uterine contraction-force continued in all pregnant groups until 10-8 mol/L concentration of Dex was added in organ baths [Figure 2b]. Whereas Dex in 10-8 mol/L increased the amplitude of uterine contraction-force in early and middle pregnancy terms of rats, it decreased the amplitude of uterine contraction-force in late-pregnancy term of rats.

The highest concentration of Dex (10-4 mol/L) decreased the amplitude of spontaneous uterine contraction-force in late-pregnancy term of rats and increased in early and middle pregnancy terms of rats by comparison with the percentage of baseline of spontaneous contraction-force [Figure 2b].

Effects of Dexmedetomidine on Contraction-Frequency

The uterine spontaneous contraction-frequency was different in an each pregnancy term (early, middle and late-pregnancy terms of rats) [Figure 3a and Table 1]. While 10-9 mol/L concentration of Dex did not affect the uterine contraction-frequency, it increased uterine contraction-frequency in early and middle-pregnancy terms in a concentration-dependent manner [Figure 3a]. After 10-9 mol/L concentration of Dex was added in organ baths, in a concentration-dependent manner, Dex decreased uterine contraction-frequency in late-pregnancy term [Figure 3a].

Figure 3

(a) Dexmedetomidine (Dex)-induced changes in calcium-free on uterine contractile-force of in pregnancy terms of pregnant rats. In calcium-free, Dex significantly was changed the contractile-force in a concentration dependent manner in all pregnancy terms (early-term (●), middle-term (○) and late-term (▼). Each point represents the percentage mean value ± SEM. *P < 0.05 as compared to control at each concentration. #P < 0.05 as compared to early-term at each concentration. +P < 0.05 as compared to early-term at each concentration, (b) Dex- induced changes in calcium-free on uterine contractile-frequency in pregnancy terms of pregnant rats. In calcium-free, Dex-induced changes the frequency of uterine contraction in all pregnancy terms (early-term (●), middle-term (○) and late-term (▼). Each point represents the percentage mean value ± SEM. *P < 0.05 as compared to control at each concentration. #P < 0.05 as compared to early-term at each concentration. +P < 0.05 as compared to early-term at each concentration

Table 1

Effect of calcium-free solution on uterine contractile activity (force and frequency) in all pregnancy term of pregnant rats

Effect of Ca2+ Free Solution on Dexmedetomidine-Induced Contraction-Frequency

While Dex caused an increase in a dose-dependent uterine contraction-frequency in middle-pregnancy term of rats in Ca2+-free [Figure 3b], it did not affect uterine contraction-frequency in late-pregnancy term of rats.

While uterine contraction-frequency increased up to 10-7 mol/L Dex in early-pregnancy term of rats, contraction-frequency severely decreased after 10-7 mol/L concentration of Dex [Table 1 and Figure 3b]. While contraction-frequency decreased up to 10-8 mol/L Dex in middle-pregnancy term of rats, contraction-frequency severely increased after 10-8 mol/L concentration of Dex.

The highest concentration of Dex (10-4 mol/L) decreased the uterine contraction-frequency in a dose-dependent manner in early and late pregnancy term of rats [Figure 3b]. However, Dex increased the uterine contraction-frequency in a dose-dependent manner in middle-pregnancy term of rats. In the presence of L-type Ca2+ channel blocker nifedipine (10-4 mol/L) in organ baths, the increasing concentration of Dex affected neither uterine contraction-force nor uterine contraction-frequency in an each pregnancy term of rats; they were not shown in this study.

Discussion

Contraction-force of smooth muscle is regulated by the cytosolic Ca2+ and the sensitivity to Ca2+ of the contractile elements in response to changes in the environment surrounding the cell. The event in regulation includes the binding of endogenous substances, such as neurotransmitters and hormones, to their specific receptors.[3] In this study, we observed that a highly selective α2 -AR agonist, Dex could directly enhance amplitude of the contraction-force and frequency of uterine rings in a dose-dependent manner.

The concentration of intracellular calcium ions constitutes the most important factor controlling force in the uterine tissue. The L-type Ca2+ channel is regulated by a wide variety of second messengers, protein kinase A, C (PKA, C), and G that are produced when agonists bind receptors on the myometrial membrane.[6] In the uterus, PKC enhances the Ca2+ current and this can be noticed as part of the source of mechanism through which agonists activate the uterus. When agonist as Dex allows release of Ca2+ from sarcoplasmic reticulum (SR) or from store-operated calcium entry (SOCE), it can increase uterine excitability. To explain alternatively, SR Ca2+ depletion activates SOCE. SOCE is well described in non-excitable cells and increasingly in smooth muscle. Furthermore, ion channel expression, Na+ channels, Ca2+ channels are altered if K+ channels and gap junctions increase in pregnancy terms. In middle pregnancy of rats, longitudinal muscle possesses mainly β-adrenoreceptors, while circular muscle has α-adrenoreceptors.[18-22] However, in late pregnancy term of rats, activation of the α-AR occurs in the longitudinal muscle while the circular muscle switches from α to β-adrenoreceptor dominance.[23] Highly selective α2 -AR agonist Dex may also influence β-adrenoreceptor dominance in middle pregnancy term that caused an increase in the amplitude of the contraction-force and frequency of uterine rings at minimal cumulative concentration (10-9 mol/L).

In contrast, it is generally accepted that in fast-striated muscles, the SR is a storage site of activator calcium.[291424] However, in smooth muscle as the uterus, contractions may be elicited even if Ca2+ entry is prevented as in these pregnancy terms. When Kreb's solution exchanged with calcium-free solution, the amplitude of contraction-force of uterine rings decreased; however, they did not disappear. If an agonist such as oxytocin or Dex is exerted to the uterus, a small transient can be increased in Ca2+ and force can occur.[25-27]

This indicates that there will be agonist-released intracellular Ca2+ store, the SR. Calcium can be released by inositol triphosphate (IP3) or calcium itself (Ca2+-induced Ca2+ release). IP3 levels increase when agonists bind to their G-coupled receptors and stimulate the hydrolysis of phosphatidylinositol. Thus, it would seem reasonable to assume that the SR Ca2+ store contributes to agonist-induced contractions.[924] Additionally, smooth muscle cells do not normally acquire fast Na+ channels. However, tetrodotoxin–sensitive Na+ channels have been found in myometrium tissue of pregnancy terms of rats and their number increased with pregnancy term, and they might cause continuation of myometrial contractions in gestation terms.

Oxytocin and prostaglandins are important myometrial agonists during pregnancy and labor, and they induce IP3 receptor activation and SR Ca2+ release that oxytocin increases in pregnancy terms. Although an increase in Ca2+ influx is a major control of smooth muscle contraction, hormones can also enhance contractile activity without directly increasing Ca2+ influx.[1227] Non-specific cation channel described with agonist stimulating in the uterus smooth muscle might cause a contribution in the contractile activity of pregnant rat uterus as Dex. In addition, the neurohypophysial hormone oxytocin is associated with pregnancy term, oxytocin increases during pregnancy term; calcium-activated chloride channels have been described in rat myometrium stimulated with oxytocin. These channels may be activated by Ca2+ release from SR. The fact that oxytocin increases Ca2+ influx more in the presence than in the absence of extracellular calcium in these cells is consistent with another form of calcium entry.[28] Capacitative or SOCE is a candidate for calcium entry distinct from voltage-operated entry.[10112930] This type of entry can be stimulated by depletion of intracellular calcium stores as a result of agonist-stimulated increases in IP3 or inhibition of intracellular calcium pumps. A number of lines of evidence point to the importance of capacitative calcium entry in myometrium.[14] Dex may cause an increase in cytosolic Ca2+ at uterus muscles. This α2-adrenergic agonist Dex may directly affect the human myometrium by enhancing the contraction-force and frequency.

Our in vitro study displayed that this effect was probably minimal at clinical doses of Dex in early and late pregnancy terms of rats; however, use of Dex should be avoided in middle pregnancy term since even the lowest dose of Dex has the potential to stimulate uterine contraction-force and contraction-frequency. During the onset of pregnancy terms, we conclude that changes in contraction-force and frequency may encounter undesirable conditions in pregnancy such as abortion and end of pregnancy. As a consequence; we need further in vitro studies as contraction mechanism and hormones levels for successful pregnancy term. After further studies, we might elucidate the true clinical effects of this agent on the myometrial activity for usefulness of Dex in the administration of anesthesia and post-operative anesthesia. Its use in parturient should therefore be avoided until additional clinical studies are performed. This study is going on to reveal contraction mechanism in pregnant rats.